asepting procesing
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BySUNILBOREDDY
M.Pharmacy
Certain pharmaceutical products must be sterile◦ injections, ophthalmic preparations, irrigations
solutions, haemodialysis solutions
Two categories of sterile products◦ those that can be sterilized in final container
(terminally sterilized)◦ those that cannot be terminally sterilized and
must be aseptically prepared
Aseptic processing Objective is to maintain the sterility of a
product, assembled from sterile components Operating conditions so as to prevent
microbial contamination
Objective To review specific issues relating to the
manufacture of aseptically prepared products:◦ Manufacturing environment
Clean areas Personnel
◦ Preparation and filtration of solutions◦ Pre-filtration bioburden◦ Filter integrity/validation◦ Equipment/container preparation and
sterilization◦ Filling Process ◦ Validation of aseptic processes◦ Specific issues relating to Isolators, BFS and Bulk
Classification of Clean Areas◦ Comparison of classifications
WHO GMP US 209E US Customary ISO/TC (209) ISO 14644
EEC GMP
Grade A M 3.5 Class 100 ISO 5 Grade A Grade B M 3.5 Class 100 ISO 5 Grade B Grade C M 5.5 Class 10 000 ISO 7 Grade C Grade D M 6.5 Class 100 000 ISO 8 Grade D
Table 1
Classification of Clean Areas◦ Classified in terms of airborne particles (Table 2)
Grade At rest In operation
maximum permitted number of particles/m3
0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm > 5 µ
A 3 500 0 3 500 0
B 3 500 0 350 000 2 000
C 350 000 2 000 3 500 000 20 000
D 3 500 000 20 000 not defined not defined
“At rest” - production equipment installed and operating
“In operation” - Installed equipment functioning in defined operating mode and specified number of personnel present
Four grades of clean areas: Grade D (equivalent to Class 100,000, ISO 8):
◦ Clean area for carrying out less critical stages in manufacture of aseptically prepared products eg. handling of components after washing.
Grade C (equivalent to Class 10,000, ISO 7):◦ Clean area for carrying out less critical stages in
manufacture of aseptically prepared products eg. preparation of solutions to be filtered.
Grade B (equivalent to Class 100, ISO 5): ◦ Background environment for Grade A zone, eg.
cleanroom in which laminar flow workstation is housed.
Grade A (equivalent to Class 100 (US Federal Standard 209E), ISO 5 (ISO 14644-1):◦ Local zone for high risk operations eg. product filling,
stopper bowls, open vials, handling sterile materials, aseptic connections, transfer of partially stoppered containers to be lyophilized.
◦ Conditions usually provided by laminar air flow workstation.
Each grade of cleanroom has specifications for viable and non-viable particles ◦ Non-viable particles are defined by the air classification
(See Table 2)
Limits for viable particles (microbiological contamination)
Grade Air sample (CFU/m3)
Settle plates (90mm diameter)
(CFU/4hours)
Contact plates (55mm
diameter) (CFU/plate)
Glove print (5 fingers)
(CFU/glove)
A < 3 < 3 < 3 < 3 B 10 5 5 5 C 100 50 25 - D 200 100 50 -
Table 3– These are average values– Individual settle plates may be exposed for less than 4 hours
• Values are for guidance only - not intended to represent specifications• Levels (limits) of detection of microbiological contamination should be established for alert and action purposes and for monitoring trends of air quality in the facility
Environmental Monitoring Physical
◦ Particulate matter
◦ Differential pressures
◦ Air changes, airflow patterns
◦ Clean up time/recovery
◦ Temperature and relative humidity
◦ Airflow velocity
Environmental Monitoring - Physical Particulate matter
◦ Particles significant because they can contaminate and also carry organisms
◦ Critical environment should be measured not more than 30cm from worksite, within airflow and during filling/closing operations
◦ Preferably a remote probe that monitors continuously◦ Difficulties when process itself generates particles (e.g.
powder filling)◦ Appropriate alert and action limits should be set and
corrective actions defined if limits exceeded
Environmental Monitoring - Physical Differential pressures
◦ Positive pressure differential of 10-15 Pascals should be maintained between adjacent rooms of different classification (with door closed)
◦ Most critical area should have the highest pressure◦ Pressures should be continuously monitored and
frequently recorded.◦ Alarms should sound if pressures deviate◦ Any deviations should be investigated and effect on
environmental quality determined
Environmental Monitoring - Physical Air Changes/Airflow patterns
◦ Air flow over critical areas should be uni-directional (laminar flow) at a velocity sufficient to sweep particles away from filling/closing area
◦ for B, C and D rooms at least 20 changes per hour are ususally required
Clean up time/recovery◦ Particulate levels for the Grade A “at rest” state
should be achieved after a short “clean-up” period of 20 minutes after completion of operations (guidance value)
◦ Particle counts for Grade A “in operation” state should be maintained whenever product or open container is exposed
Environmental Monitoring - Physical Temperature and Relative Humidity
◦ Ambient temperature and humidity should not be uncomfortably high (could cause operators to generate particles) (18°C)
Airflow velocity◦ Laminar airflow workstation air speed of approx
0.45m/s ± 20% at working position (guidance value)
Personnel Minimum number of personnel in clean areas
◦ especially during aseptic processing Inspections and controls from outside Training to all including cleaning and
maintenance staff◦ initial and regular◦ manufacturing, hygiene, microbiology◦ should be formally validated and authorized to enter
aseptic area Special cases
◦ supervision in case of outside staff◦ decontamination procedures (e.g. staff who worked
with animal tissue materials)
Personnel (2) High standards of hygiene and cleanliness
◦ should not enter clean rooms if ill or with open wounds
Periodic health checks No shedding of particles, movement slow and
controlled No introduction of microbiological hazards No outdoor clothing brought into clean areas,
should be clad in factory clothing Changing and washing procedure No watches, jewellery and cosmetics Eye checks if involved in visual inspection
Personnel (3) Clothing of appropriate quality:
◦ Grade D hair, beard, moustache covered protective clothing and shoes
◦ Grade C hair, beard, moustache covered single or 2-piece suit (covering wrists, high
neck), shoes/overshoes no fibres/particles to be shed
◦ Grade A and B headgear, beard and moustache covered,
masks, gloves not shedding fibres, and retain particles shed
by operators
Personnel (4) Outdoor clothing not in change rooms leading to
Grade B and C rooms Change at every working session, or once a day (if
supportive data) Change gloves and masks at every working session Frequent disinfection of gloves during operations Washing of garments – separate laundry facility
◦ No damage, and according to validated procedures (washing and sterilization)
Regular microbiological monitoring of operators
In aseptic processing, each component is individually sterilised, or several components are combined with the resulting mixture sterilized.◦ Most common is preparation of a solution which is
filtered through a sterilizing filter then filled into sterile containers (e.g active and excipients dissolved in Water for Injection)
◦ May involve aseptic compounding of previously sterilized components which is filled into sterile containers
◦ May involve filling of previously sterilized powder sterilized by dry heat/irradiation produced from a sterile filtered solution which is then
aseptically crystallized and precipitated requires more handling and manipulation with higher potential
for contamination during processing
Preparation and Filtration of Solutions Solutions to be sterile filtered prepared in a Grade
C environment If not to be filtered, preparation should be
prepared in a Grade A environment with Grade B background (e.g. ointments, creams, suspensions and emulsions)
Prepared solutions filtered through a sterile 0.22μm (or less) membrane filter into a previously sterilized container◦ filters remove bacteria and moulds◦ do not remove all viruses or mycoplasmas
filtration should be carried out under positive pressure
Preparation and Filtration of Solutions (2) consideration should be given to complementing
filtration process with some form of heat treatment Double filter or second filter at point of fill
advisable Fitlers should not shed particles, asbestos
containing filters should not be used Same filter should not be used for more than one
day unless validated If bulk product is stored in sealed vessels, pressure
release outlets should have hydrophobic microbial retentive air filters
Preparation and Filtration of Solutions (3) Time limits should be established for each phase of
processing, e.g.◦ maximum period between start of bulk product
compounding and sterilization (filtration)◦ maximum permitted holding time of bulk if held after
filtration prior to filling◦ product exposure on processing line◦ storage of sterilized containers/components◦ total time for product filtration to prevent organisms from
penetrating filter◦ maximum time for upstream filters used for clarification
or particle removal (can support microbial attachment)
Preparation and Filtration of Solutions (4) Filling of solution may be followed by lyophilization
(freeze drying)◦ stoppers partially seated, product transferred to
lyophilizer (Grade A/B conditions)◦ Release of air/nitrogen into lyophilizer chamber at
completion of process should be through sterilizing filter
Prefiltration Bioburden (natural microbial load) Limits should be stated and testing should be carried
out on each batch Frequency may be reduced after satisfactory history is
established◦ and biobuden testing performed on components
Should include action and alert limits (usually differ by a factor of 10) and action taken if limits are exceeded
Limits should reasonably reflect bioburden routinely achieved
Prefiltation Bioburden (2) No defined “maximum” limit but the limit should not
exceed the validated retention capability of the filter Bioburden controls should also be included in “in-
process” controls ◦ particularly when product supports microbial growth
and/or manufacturing process involves use of culture media
Excessive bioburden can have adverse effect on the quality of the product and cause excessive levels of endotoxins/pyrogens
Filter integrity Filters of 0.22μm or less should be used for filtration
of liquids and gasses (if applicable)◦ filters for gasses that may be used for purging or
overlaying of filled containers or to release vacuum in lyphilization chamber
filter intergrity shoud be verified before filtration and confirmed after filtration◦ bubble point◦ pressure hold◦ forward flow
methods are defined by filter manufacturers and limits determined during filter validation
Equipment/container preparation and sterilization
All equipment (including lyophilizers) and product containers/closures should be sterilized using validated cycles◦ same requirements apply for equipment sterilization
that apply to terminally sterilized product◦ particular attention to stoppers - should not be tightly
packed as may clump together and affect air removal during vacuum stage of sterilization process
◦ equipment wrapped and loaded to facilitate air removal
◦ particular attention to filters, housings and tubing
Equipment/container preparation and sterilization (2)
CIP/SIP processes◦ particular attention to deadlegs - different orientation
requirements for CIP and SIP heat tunnels often used for
sterilization/depyrogenation of glass vials/bottles◦ usually high temperature for short period of time◦ need to consider speed of conveyor◦ validation of depyrogenation (3 logs endotoxin units)
worst case locations◦ tunnel supplied with HEPA filtered air
Equipment/container preparation and sterilization (2)
equipment should be designed to be easily assembled and disassembled, cleaned, sanitised and/or sterilized◦ equipment should be appropriately cleaned - O-rings and gaskets
should be removed to prevent build up of dirt or residues rinse water should be WFI grade equipment should be left dry unless sterilized immediately
after cleaning (to prevent build up of pyrogens) washing of glass containers and rubber stoppers should be
validated for endotoxin removal should be defined storage period between sterilization and
use (period should be justified)
Additional issues specific to Isolator and BFS Technologies
Isolators◦ Decontamination process requires a 4-6 log
reduction of appropriate Biological Indicator (BI)◦ Minimum 6 log reduction of BI if surface is to be free
of viable organisms◦ Significant focus on glove integrity - daily checks,
second pair of gloves inside isolator glove◦ Traditional aseptic vigilance should be maintained
Blow-Fill-Seal (BFS)◦ Located in a Grade D environment◦ Critial zone should meet Grade A (microbiological)
requirements (particle count requirements may be difficult to meet in operation)
◦ Operators meet Grade C garment requirements◦ Validation of extrusion process should demonstrate
destruction of endotoxin and spore challenges in the polymeric material
◦ Final inspection should be capable of detecting leakers
Issues relating to Aseptic Bulk Processing• Applies to products which can not be filtered at point of
fill and require aseptic processing throughout entire manufacturing process.
• Entire aseptic process should be subject to process simulation studies under worst case conditions (maximum duration of "open" operations, maximum no of operators)
• Process simulations should incorporate storage and transport of bulk.
• Multiple uses of the same bulk with storage in between should also be included in process simulations
• Assurance of bulk vessel integrity for specified holding times.
Bulk Processing (2)• Process simulation for formulation stage should be
performed at least twice per year.◦ Cellular therapies, cell derived products etc
products released before results of sterility tests known (also TPNs, radioactive preps, cytotoxics)
should be manufactured in a closed system Additional testing
sterility testing of intermediates microscopic examination (e.g. gram stain) endotoxin testing
Environmental Monitoring Environmental Monitoring ConsiderationsConsiderations
Airborne nonviable particulate monitoring Airborne viable contaminant monitoring Viable contaminant monitoring of surfaces Viable contaminant monitoring of personnel Temperature and humidity monitoring Pressure differential monitoring
Environmental Monitoring Environmental Monitoring ComponentsComponents
Water monitoring:◦ Total organic carbon◦ Conductivity◦ Microbial Contaminants◦ Endotoxin
Environmental Monitoring Environmental Monitoring ComponentsComponents
Monitoring frequencies and strategies◦ Establishment of a meaningful and manageable
program Sampling and testing procedures Establishment of effective alert and action
limits Trending of results
General Environmental General Environmental Monitoring ConsiderationsMonitoring Considerations
Investigation and evaluation of trends as well as excursions from alert and action limits
Corrective actions to be implemented in response to environmental monitoring excursions
Personnel training - sampling, testing, investigating excursions, aseptic technique
General Environmental General Environmental Monitoring ConsiderationsMonitoring Considerations
Should include monitoring of all environments where products and their components are manufactured◦ All areas where there is a risk of product
contamination Should include monitoring of all water used
for product manufacturing as well as feed water to the final water purification system (WFI System)
Scope of Environmental Scope of Environmental Monitoring ProgramMonitoring Program
CFR GMP regulations FDA Guidance Documents USP Informational Chapter
Regulatory Basis for Regulatory Basis for Environmental Monitoring Environmental Monitoring ProgramProgram
Aseptic processing areas:◦ Easy to clean and maintain◦ Temperature and humidity controlled◦ HEPA filtered air◦ Environmental monitoring system◦ Cleaning and disinfecting procedures◦ Scheduled equipment maintenance and
calibration
21 CFR 211.4221 CFR 211.42
Ventilation, air filtration, air heating and cooling:◦ Adequate control over microorganisms, dust,
humidity and temperature.◦ Air filtration systems including prefilters and
particulate matter air filters for air supplies to production areas.
21 CFR 211.4621 CFR 211.46
Defines critical and controlled manufacturing areas
Recommends airborne nonviable and viable contaminant limits
Provides some guidance on monitoring frequencies for critical areas
Guideline on Sterile Drug Guideline on Sterile Drug Products Produced by Aseptic Products Produced by Aseptic ProcessingProcessing
Recommendations for air pressure differentials
Includes guidance on aseptic media fills Note: This guidance document was written
in 1987 and is in need of revision
Guideline on Sterile Drug Guideline on Sterile Drug Products Produced by Aseptic Products Produced by Aseptic ProcessingProcessing
USP General Information Chapter <1116> Establishment of clean room classifications
◦ Federal Standard 209E Importance of EM program Personnel training in aseptic processing Establishment of sampling plans and sites
◦ suggested sampling frequencies
Microbial Evaluation and Microbial Evaluation and Classification of Clean Rooms and Classification of Clean Rooms and Clean ZonesClean Zones
Establishment of alert and action limits Suggests limits for airborne, surface and
personnel contaminant levels. Methods and equipment for sampling Identification of isolates Aseptic media fills Emerging technologies - barrier; isolator
Microbial Evaluation and Microbial Evaluation and Classification of Clean Rooms and Classification of Clean Rooms and Clean ZonesClean Zones
“Airborne Particulate Cleanliness Classes in Clean Rooms and Clean Zones
Approved by the GSA for use by all Federal Agencies
Frequently referenced for controlled environment particulate requirements: Classes 100, 10,000 and 100,000 (based on particles > 0.5µ)
Federal Standard 209EFederal Standard 209E
Scope limited to final drug product manufacturing and data required for application submission (NDA, BLA)
Requests information on:◦ Buildings and facilities◦ Manufacturing operations for drug product Filter validation Validation of hold times
Guidance for Industry for Sterile Guidance for Industry for Sterile Validation Process Validation in Validation Process Validation in Applications for Human and Veterinary Applications for Human and Veterinary Drug ProductsDrug Products
Requests information on:◦ Sterilization and depyrogenation◦ Media fills and actions taken when they fail◦ Microbiological monitoring of the environment
Airborne microorganisms, personnel, surfaces, water system, product component bioburden
◦ Yeasts, molds, anaerobes◦ Exceeded EM limits
Guidance for Industry for Sterile Guidance for Industry for Sterile Validation Process Validation in Validation Process Validation in Applications for Human and Veterinary Applications for Human and Veterinary Drug ProductsDrug Products
Viable and Nonviable Viable and Nonviable Contaminant LimitsContaminant Limits
Classifi-cation
Nonviable (>0.5µ) Viable
ft3 m3 ft3 m3
Class 100
100 3,530 0.1 3.5
Class 10,000
10,000 353,000 0.5 18
Class 100,000
100,000 3,530,000 2.5 88
Preparation or manufacturing area where nonsterile product, in-process materials and product-contact equipment surfaces, containers and closures are exposed to the environment
Control nonviable and viable contaminants to reduce product /process bioburden
Class 100,000 or Class 10,000
Controlled AreaControlled Area
Capping areas are now considered controlled manufacturing areas◦ Should be supplied with HEPA filtered air◦ Should meet class 100,000 conditions during
static conditions
Controlled AreaControlled Area
Aseptic processing area where sterile products, components or in-process products are exposed to the environment and no further processing will occur.
Air quality must be Class 100 during processing
Local Class 100 areas are often utilized during open processing steps during drug substance manufacture.
Critical AreaCritical Area
The area just preceding the sterile core should be one classification higher than the core.
Critical AreaCritical Area
Airborne cleanliness classifications should be met during operations
Nonviable monitoring should occur routinely during operations
Monitoring during static conditions is done as part of HVAC qualification and may be done periodically after that to insure area meets acceptable conditions before use or following cleaning
Nonviable Particulate Nonviable Particulate MonitoringMonitoring
Locations for monitoring should be established during performance qualification; probes placed close to work surface
Monitoring frequencies vary:◦ For aseptic processing areas, during each use◦ For other, controlled areas, varies from each use
to weekly or less depending on use of area
Nonviable Particulate Nonviable Particulate MonitoringMonitoring
HVAC Validation and Maintenance Considerations:◦ Air velocity, airflow patterns and turbulence
should be validated; smoke studies to determine flow patterns during static and dynamic conditions
◦ HEPA filter integrity testing ◦ HEPA filter efficiency testing◦ Air pressure differentials
Nonviable Particulate Nonviable Particulate MonitoringMonitoring
Airborne viable contaminants Surface contaminants
◦ walls◦ equipment surfaces◦ countertops◦ floors
Personnel contaminants
Microbial MonitoringMicrobial Monitoring
Monitoring methods should be capable of detecting molds and yeasts
Should also be able to detect anaerobes◦ Most often, this is an issue associated with
products filled anaerobically (with nitrogen overlay)
All lots of media for EM sampling should be growth promotion tested
Microbial MonitoringMicrobial Monitoring
Routine microbial monitoring should take place during operations (for airborne contaminants) and immediately following operations (for surfaces and personnel).
Airborne monitoring frequencies:◦ Each use for aseptic processing areas◦ Varies from daily to weekly to less frequently for
controlled areas depending on use
Microbial MonitoringMicrobial Monitoring
Personnel and surface monitoring frequencies vary:◦ Aseptic processing - after every fill◦ Other controlled areas - varies from daily to
weekly or less for surfaces ◦ Personnel monitoring often restricted to aseptic
area personnel and personnel working in Class 100 hoods performing tasks such as inoculation
Microbial MonitoringMicrobial Monitoring
Monitoring of surfaces and airborne contaminants during rest periods (following cleaning)◦ Important for confirming adequacy of cleaning
procedures◦ Indicates whether HVAC system is operating
properly◦ NOTE: Disinfectant effectiveness studies also
required for cleaning agents used in the facility
Microbial MonitoringMicrobial Monitoring
Monitoring frequencies and procedures are influenced by a number of factors:◦ Stage of manufacturing ◦ “Open” or “closed” manufacturing step◦ Single or multiple product manufacturing
Microbial MonitoringMicrobial Monitoring
Establishment of monitoring locations should be based on performance qualification studies during dynamic conditions◦ gridding study to determine worst case
locations/most meaningful locations Should also establish common flora - will aid
in investigations
Microbial MonitoringMicrobial Monitoring
Action limits (for the most part) have been established in a variety of guidance documents
Alert limits ◦ Lower than action limits ◦ Reflect actual historical results under normal
processing conditions
Setting Alert and Action Setting Alert and Action LimitsLimits
Alert limits are designed to provide some warning that environmental quality is approaching action limit and allow you time to correct.
Exceeding alert limit triggers a warning response - i.e., alert affected area personnel
Exceeding multiple alerts - triggers action level response
Exceeding LimitsExceeding Limits
Action limit excursions require investigations◦ Speciation of organism(s)◦ Review batch records from date of excursion◦ Review other recent EM data (trends)◦ Review cleaning records◦ Interview personnel◦ Product impact - must quarantine until
determined
Exceeding LimitsExceeding Limits
Excursions from action limits require corrective actions that may include:◦ More rigorous or additional monitoring◦ More rigorous cleaning◦ Retraining of personnel◦ Procedural changes - change to or addition of
disinfection procedures, for example◦ HVAC maintenance
Exceeding LimitsExceeding Limits
The investigation procedures to be followed should be pre-established and included in SOPs
Depending on the outcome of the investigation, corrective actions should be pre-established to the extent possible
Investigations and Corrective Investigations and Corrective ActionsActions
Imperative that EM results be linked to product release so that affected products are not released until investigation completed
Material Review Board or equivalent should be consulted prior to releasing product that was potentially affected by adverse environmental conditions
Investigations and Corrective Investigations and Corrective ActionsActions
Should trend monitoring results (environmental and water) ◦ Periodic (quarterly or monthly) review by QA and
others◦ Re-evaluation of action and alert limits on an
annual basis◦ This trending information is generally included in
the Annual Product Review
TrendingTrending
Control of temperature and humidity required for aseptic processing areas ◦ 21 CFR 211.42(c)(10)(ii)
Generally 65°F and 35-50% humidity are average◦ Too high - Increases personnel shedding◦ Too low - Increase static electricity
Temperature and HumidityTemperature and Humidity
Temperature should be controlled throughout all manufacturing areas
Temperature and humidity should be monitored and controlled in warehouse areas where temperature/humidity sensitive raw materials are stored◦ If not able to control humidity, need procedure to
follow if humidity exceeds limit
Temperature and HumidityTemperature and Humidity
Water RequirementsWater Requirements
Test Potable Water
Purified Water
WFI
TOC none 500 ppb 500 ppb
Conduc-tivity
none See USP Table
Micro. Purity
500 CFU/ml
100 CFU/ml
10 CFU/ 100 ml
Endo- Toxin
none none 0.25 EU/ml
Water purified by distillation or reverse osmosis
Prepared from water complying with the U.S. EPA National Primary Drinking Water Regulations
Contains no added substance
Water For InjectionWater For Injection
Obtained by a suitable process, usually one of the following:◦ deionization◦ reverse osmosis◦ combination
Purified WaterPurified Water
Meets National Drinking Water Regulations 40 CFR Part 141 Periodic monitoring in-house as well as
periodic certificates from municipality (if applicable)
Potable WaterPotable Water
WFI Systems◦ Microbial quality and endotoxin
Daily system monitoring Each use point at least weekly
◦ TOC and Conductivity Weekly system monitoring can be taken from worst case point (end of loop,
return to tank)
Water System MonitoringWater System Monitoring
Purified Water Systems◦ Weekly monitoring of system for: microbial quality TOC conductivity
Water System MonitoringWater System Monitoring
WFI ◦ Solvent for preparation of parenteral solutions◦ Formulation of mammalian cell culture media◦ Formulation of purification buffers◦ Final product formulation◦ Vial and stopper washing ◦ Final rinse for product equipment
Water UseWater Use
Purified Water◦ Preparation of terminally sterilized microbiological
media◦ Initial rinsing/cleaning◦ Laboratory use◦ Feed for WFI system
Water UseWater Use
Potable Water◦ Non-product contact uses◦ Feed for purified water system
Water UseWater Use
Slit-to-Agar (STA) - Powered by vacuum, air taken in through a slit below which is a slowly revolving plate.
Sieve impactor - Vacuum draws in air through perforated cover which is impacted onto petri dish containing nutrient agar
Microbial Monitoring Microbial Monitoring DevicesDevices
Centrifugal Sampler - consists of a propeller that pulls a known volume of air into the unit and then propels the air outward to impact on a nutrient agar strip
Sterilizable Microbiological Atrium (SMA)- similar to sieve impactor; cover contains uniformly spaced orifices; vacuum draws in air which is impacted on agar plate
Microbial Monitoring Microbial Monitoring DevicesDevices
Surface Air System Sampler - An integrated unit containing an entry section with an agar contact plate; behind is a motor and turbine that pulls air in through the perforated cover and exhausts it beyond the motor.
Settle plates - qualitative; may be useful in worst case locations
Microbial Monitoring Microbial Monitoring DevicesDevices
Surface contaminant monitoring devices:◦ Contact Plates - plates filled with nutrient agar;
for regular surfaces◦ Swabs - useful for hard to reach or irregular
surfaces; swab placed in suitable diluent and inoculated onto microbiological plate
Microbial Monitoring Microbial Monitoring DevicesDevices
Remote sampling probes - validate use of tubing
Must sample adequate quantity of air to be statistically meaningful.◦ 80-100 ft3/min
Must validate growth promotion after exposure of settle plates (or other plates) for prolonged time periods.
Monitoring ConsiderationsMonitoring Considerations
Contamination Control
Methods to Achieve Methods to Achieve CleanlinessCleanliness Positive Pressure / Airflow
◦ Keeps contamination out of the work area◦ Depends on clean air input
Filtration◦ Development of effective filtration revolutionized industry◦ HEPA (High Efficiency Particulate Air) and ULPA (Ultra Low
Particulate Air) Filters Materials Selection User Protocols Cleaning
Facility DesignFacility Design
Complete cleanroom created with centralized air handling or fan filter units
Keeps entire room clean Requires complete gowning, careful
materials and equipment selection to maintain class
Costly, often unnecessary
Facility DesignFacility Design
Can use localized clean areas Clean Benches: Horizontal and Vertical
Laminar Flow (HLF on left, VLF on right)
Facility designFacility design
Isolators, Glove boxes provide better protection from outside contamination
Contamination Control by Contamination Control by LayoutLayout Isolation between processes
prevents cross contamination; separate rooms, air showers, door interlocks
“Onion” concept: cleanest areas are inside, have to pass through successively cleaner areas to reach these areas
Air Flow & TurbulenceAir Flow & Turbulence
Most airflow is turbulent—no clear relation between velocity vectors at different points
•Particles can be trapped in eddies for long time•Not optimal for contamination control!! Long path length for contamination to leave the room
Laminar (Unidirectional) Laminar (Unidirectional) Air FlowAir Flow
Concept of laminar airflow
In cleanrooms, often called uni-directional flow (UDF)
• Ideal for contamination control—shortest path to sweep particles out of clean areas; complete room air change in shortest period of time
High level cleanrooms designed for laminar flow in most areasCost means that for most, clean areas are some combination of laminar and turbulent flowNot always a simple tradeoff—with turbulent flow, require higher air velocities, which require larger air handlers.
UDF More Important for UDF More Important for Cleaner AreasCleaner Areas
Practical Considerations Practical Considerations for UDFfor UDF Any objects in path of laminar flow will
deflect airflow—this usually results in turbulence; USER BEHAVIOR HAS LARGE IMPACT•Most critical for laminar flow benches situated in non-clean areas; not as critical if located in larger clean area
Types of ContaminationTypes of Contamination
Particulate—encompasses most contamination
Chemical—films, vapors, etc. Biological—bacteria, viruses, etc.; for our
purposes, treat as particles Similar concerns for rooms & equipment
as for substrates
Airborne ContaminationAirborne Contamination
From Cleanrooms Magazine, 2000
Invisible to naked eye below ~50um without special illumination
Particulate Particulate ContaminationContamination Biggest concern for LCI cleanroom users Basis for classification of cleanrooms Does include biological contamination as
a subset of total particulates Many sources: personnel, equipment, etc.
Microbial ContaminationMicrobial Contamination
Outer layer of human skin can host up to 1 million microorganisms per square cm
Human saliva up to 1 billion per mL Bacteria is usually primary concern, but
foreign organic matter, viruses, fungi, algae are all included here
Cross contamination can be a big problem.
Contamination Contamination MeasurementMeasurement Particulate contamination typically
measured with laser particle counter Microbial contamination can be
measured in several ways◦ Centrifugal sampler◦ Settle plate method◦ Contact plate method◦ Swabbing
Usage of MeasurementsUsage of Measurements
Complementary to yield tracking Can use measurements to isolate
problem areas Regular measurements can help to
track changes, which can then be tied back to protocol, personnel, or material changes◦ Don’t depend upon room to maintain itself.
RealityReality
In a perfect world, could monitor many points on a very regular basis
In reality, this is usually not practical, due to personnel time and financial constraints
Important to identify a realistic test & measurement program
Contamination Control Contamination Control and Its Relationships and Its Relationships All sources of contamination and control
are interrelated
CleaningCleaning
Critical to remove contaminants that cannot be removed by air handling
Important to follow procedures appropriate to your application
What is appropriate for one industry may not be appropriate for another
Most important thing is to develop standard procedures and FOLLOW THEM
Surfaces are importantSurfaces are important
The efficiency of these cleaning methods depends on the surface being cleaned
Rough or pitted surfaces are more difficult to clean
Sharp corners are difficult to clean That’s why inside surfaces of clean rooms
are smooth.
VacuumingVacuuming
Dry and wet◦ Dry has low (<25% ) efficiency for particles
smaller than 10 microns (about .0005 inches)◦ wet uses liquids which result in greater force
on the particles and hence better cleaning
Wet wipingWet wiping
Can be very efficient Liquid breaks some bonds between
surface and particles and allows particles to float off
Those adhering on surface can be rubbed off and retained in wiper.
Must be careful not to redeposit particles Efficiency varies
Tacky rollersTacky rollers
Efficiency depends of tackiness of roller, cleanliness of tacky surface and softness of roller are also very important
Cleaning liquidsCleaning liquids
No ideal cleaning liquid Most facilities use DI water or isopropyl
alcohol with disinfectant Water with surfactant and disinfectant
may be used as well as alcohol-water solutions
The choice depends on what works, cost, history, etc.
Materials SelectionMaterials Selection
Choice of materials for supplies, equipment, gowning, etc. is important
“Clean” materials can become dirty!! Look for easy-to-clean materials Triboelectricity can cause static
problems, as can low humidity—this exacerbates contamination problems
Biofilms!!
General RequirementsGeneral Requirements
Minimize sources of contaminants◦ No smoking◦ No cosmetics◦ Avoid high particulate clothing, such as wool
sweaters◦ Cover up! Uncovered skin can lead to more
contamination
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